Monette M. Cotreau

Dose‐Dependent Pharmacokinetics and Psychomotor Effects of Caffeine in Humans

Twelve healthy volunteers received oral placebo, 250 mg of caffeine, and 500 mg of caffeine in a randomized, double‐blind, single‐dose crossover study. Caffeine kinetics were nonlinear, with clearance significantly reduced and elimination half‐life prolonged at the 500‐mg compared to the 250‐mg dose. The lower dose of caffeine produced more favorable subjective effects than the higher dose (elation, peacefulness, pleasantness), whereas unpleasant effects (tension, nervousness, anxiety, excitement, irritability, nausea, palpitations, restlessness) following the 500‐mg dose exceeded those of the 250‐mg dose. The lower dose of caffeine enhanced performance on the digit symbol substitution test and a tapping speed test compared to placebo; high‐dose caffeine produced less performance enhancement than the lower dose. The plasma concentration versus response relationship revealed concentration‐dependent increases in anxiety and improvements in cognitive and motor performance at low to intermediate concentrations. Both caffeine doses reduced electroencephalographic amplitude over the 4 Hz to 30 Hz spectrum, as well as in the alpha (8–11 Hz) and beta (12–30 Hz) ranges; however, effects were not dose‐dependent. While favorable subjective and performance‐enhancing stimulant effects occur at low to intermediate caffeine doses, the unfavorable subjective and somatic effects, as well as performance disruption, from high doses of caffeine may intrinsically limit the doses of caffeine used in the general population.

The influence of age and sex on the clearance of cytochrome P450 3A substrates.

Cytochrome P450s (CYPs) are an important family of enzymes in the metabolism of many therapeutic agents and endogenous metabolic reactions. The CYP3A subfamily is especially prominent in these metabolic activities. This review article focuses on how the factors of age and sex may influence the in vivo activity of human CYP3A. The functional activity of CYP3A varies based on issues such as interaction with one or more substrates and between individuals and/or localisation. For CYP3A substrates, intrinsic clearance is the component of total clearance that is contributed by the enzymes. Depending on the route of administration and the contribution of hepatic blood flow to overall clearance, sensitivities to changes in CYP3A activities may differ. Additionally, age may influence the hepatic blood flow and, in turn, affect CYP3A activity. A review of the literature regarding age influences on the clearance of CYP3A substrates does suggest that age can affect the clearance of certain CYP3A substrates.CYP3A is responsible for a large number of endogenous metabolic reactions involving steroid hormones, and enzyme activity has been reported to be induced and/or inhibited in the presence of some sex steroids. Based on published studies for most CYP3A substrates, sex does not appear to influence clearance; however, with certain substrates significant sex-related differences are found. In such cases, women primarily have higher clearance than men.

Fluoxetine impairs clearance of alprazolam but not of clonazepam

Volunteer male subjects received single 1.0 mg oral doses of alprazolam and of clonazepam on two occasions, during coadministration of 40 mg/day fluoxetine or of placebo. When the sequence of trials was placebo first and fluoxetine second, fluoxetine coadministration significantly prolonged alprazolam half‐life (20 versus 17 hours) and reduced clearance (48 versus 61 ml/min). No effect of fluoxetine was seen when fluoxetine was given first and placebo second, because norfluoxetine persisted into the placebo phase even though fluoxetine had been discontinued 2 weeks earlier. Fluoxetine had no significant effects on clonazepam elimination half‐life or clearance regardless of the sequence of fluoxetine and placebo administration. In the fluoxetine‐placebo sequence, fluoxetine significantly increased the rate of clonazepam absorption. Thus fluoxetine appears to impair clearance of alprazolam by way of microsomal oxidation but does not alter clearance of clonazepam by way of nitroreduction. The very slow elimination of norfluoxetine should be considered in the design of clinical or pharmacokinetic studies that involve fluoxetine.

The antiparasitic moxidectin: safety, tolerability, and pharmacokinetics in humans.

A study in healthy male volunteers was completed to evaluate the safety, tolerability, and pharmacokinetics of a single oral dose of the antiparasitic moxidectin (MOX). This drug is registered worldwide as a veterinary antiparasitic agent for use in companion and farm animals. This is the first study of MOX in humans. All subjects were between the ages of 18 and 45 years, with normal cardiac, hematologic, hepatic, and renal function. Doses of MOX studied were 3, 9, 18, and 36 mg in cohorts of 6 subjects each (5:1, MOX:placebo). At the 9‐mg and 36‐mg doses, two separate cohorts were completed, one in the fasted state and one after the consumption of a high‐fat breakfast. For all other cohorts, administration was in the fasted state. Safety and tolerability were assessed by physical examinations, ongoing evaluation of adverse events (AEs), and measurement of laboratory values. Pharmacokinetic (PK) samples were collected just prior to dosing and at various time points until 80 days postdose. Safety assessments from all dose groups studied suggested that MOX was generally safe and well tolerated, with a slightly higher incidence of transient, mild, and moderate central nervous system AEs as the dose increased as compared to placebo. The PKs of MOX were dose proportional within the dose range studied, and the elimination half‐life (t1/2 elim) was long (mean: 20.2–35.1 days). At the 9‐mg and 36‐mg doses, a high‐fat breakfast was shown to delay and increase the overall absorption but did not increase maximal concentrations when compared to administration in the fasted state. In summary, the results from this study indicate that MOX is safe and well tolerated in humans between the doses of 3 mg and 36 mg.

Biologic and Clinical Activity of Tivozanib (AV-951, KRN-951), a Selective Inhibitor of VEGF Receptor-1, -2, and -3 Tyrosine Kinases, in a 4-Week-On, 2-Week-Off Schedule in Patients with Advanced Solid Tumors

Purpose: To assess the maximum tolerated dose (MTD)/dose-limiting toxicities (DLT), safety, pharmacokinetics, and pharmacodynamics of tivozanib, a potent and selective oral VEGF receptor (VEGFR) tyrosine kinase inhibitor. Experimental Design: Dose levels of 1.0, 1.5, and 2.0 mg/d tivozanib for 28 days followed by 14 days of medication were explored in patients with advanced solid tumors. Results: Forty-one patients were enrolled. Animal data incorrectly predicted toxicity, resulting in DLTs at the starting dose (2.0 mg) consisting of grade 3 proteinuria and hypertension and grade 3 ataxia. At 1.0 mg, no DLT was observed. At an intermediate dose (1.5 mg), 1 patient experienced DLT consisting of grade 3 hypertension. This dose was determined as the MTD. Of 10 additional patients treated at 1.5 mg, 1 patient each experienced grade 3 hypertension and grade 3 fatigue, and 2 patients experienced grade 3 and 4 transaminase elevation. In 12 additional patients treated at 1.0 mg, no DLT was observed. Pharmacokinetics displayed long absorption time, dose proportional exposure, and a half-life of 4.7 days. Plasma levels of VEGF-A and soluble VEGFR-2 showed dose-dependent increases and decreases, respectively. Dynamic contrast-enhanced MRI indicated reduction in tumor perfusion. Clinical activity was observed in renal cell cancer, colorectal cancer, and other tumors. Conclusion: Tivozanib was well tolerated with manageable side effects. The pharmacokinetics profile revealed that tivozanib was suitable for once-daily dosing. Encouraging and durable clinical activity was observed. The recommended daily dose of tivozanib in a 4-week-on and 2-week-off dosing regimen is 1.5 mg. Clin Cancer Res; 17(22); 7156–63. ©2011 AACR.

Methodologies to study the induction of rat hepatic and intestinal cytochrome P450 3A at the mRNA, protein, and catalytic activity level

Studies were conducted to characterize assays for the isolation and quantitation of rat cytochrome P450 (CYP) 3A isoforms from hepatic and intestinal tissues. Isolated intestinal microsomes were analyzed for their alkaline phosphatase activity and CYP 3A immunoreactivity. The involvement of CYP 3A in the in vitro hydroxylation of midazolam (MDZ) was also evaluated using isoform specific chemical and antibody inhibitors. The effect of glycerol (a common constituent of the microsomal reconstitution buffer) concentration on in vitro MDZ hydroxylation was also investigated. Additionally, to verify that the intestinal preparation was adequate for use in studies investigating the induction of CYP3A at the MRNA, protein, and catalytic activity within a single animal, a separate induction study was carried out with the CYP 3A inducer dexamethasone (DEX). A reverse transcription-polymerase chain reaction (RT-PCR) assay and a quantitative Western blotting method were used to reliably detect differences in CYP 3A mRNA and immunoreactivity between DEX- and vehicle (VH)-treated tissues. The in vitro hydroxylation of MDZ evaluated CYP 3A catalytic activity and identified increases in CYP 3A activity caused by DEX in comparison to VH. Collectively, these described techniques provide an experimental model to study xenobiotic induction of rat hepatic and intestinal CYP 3A from the molecular to the catalytic level in individual rats without the need for pooling of tissue.

Unchanged cytochrome P450 3A (CYP3A) expression and metabolism of midazolam, triazolam, and dexamethasone in mdr(−/−) mouse liver microsomes

P-Glycoprotein (P-gp) and cytochrome P450 3A (CYP3A) share common substrates and expression properties, but the relationship of mdrl deficiency to CYP3A-mediated metabolism and protein expression is not established. The in vitro kinetic parameters of CYP3A-mediated metabolism of midazolam (MDZ), triazolam (TRZ), and dexamethasone (DEX) were studied in liver microsomes from three mrdrla(-/-) mice, one mdrla/b(-/-) mouse, and mdrla/b(+/+) controls. The kinetic profiles of CYP3A-mediated MDZ 4-hydroxylation were not significantly different between mdrl-deficient animals and controls. Overall mean (+/- SEM, N = 8) values were: Vmax, 0.74+/-0.05 nmol/min/mg protein; Km, 28.2+/-2.7 microM; and estimated intrinsic clearance, 0.026+/-0.003 mL/min/mg protein. Likewise, rates of formation of alpha-OH- and 4-OH-TRZ (from 500 microM TRZ), and of DEX metabolites sensitive to ketoconazole inhibition, M1 and M5 (from 20 microM DEX), did not differ between mdrl-deficient and control animals. Immunoquantified microsomal CYP3A protein levels in mdrla(-/-), mdrla/b(-/-), and mdrla/b(+/+) mice were not different, with overall mean immunoreactive protein levels of 2.68+/-0.09 pmol/microg protein. Although CYP3A and P-gp share aspects of activity and expression, disruption of the mdrl genes does not affect CYP3A-mediated metabolism or protein expression in the mouse.

Multiple‐Dose, Safety, Pharmacokinetics, and Pharmacodynamics of a New Selective Estrogen Receptor Modulator, ERA‐923, in Healthy Postmenopausal Women

ERA‐923 is a new selective estrogen receptor modulator under clinical investigation for use in tamoxifen refractory metastatic breast cancer. This study evaluated the safety, tolerability, pharmacokinetics, and pharmacodynamics of once‐daily oral ERA‐923 (10–200 mg) for 28 days in healthy postmenopausal females. ERA‐923 was well tolerated, and adverse events were mild and reversible. No clinically significant changes in laboratory values were found with ERA‐923 versus placebo. ERA‐923 appeared to undergo extensive metabolism and enterohepatic recirculation. In addition, pharmacokinetic analysis showed that a high‐fat breakfast increased the extent of absorption. ERA‐923‐dosed subjects had no uterine or ovarian changes when evaluated with transvaginal ultrasound and compared to placebo subjects. Overall, ERA‐923 was safe and well tolerated in postmenopausal women dosed for 28 days.

Molecular and pharmacokinetic evaluation of rat hepatic and gastrointestinal cytochrome P450 induction by tamoxifen

Tamoxifen (TAM) is a first-line endocrine treatment for all stages of postmenopausal breast cancer. The cytochrome P450 (CYP) enzymes catalyze the majority of TAM’s primary metabolism, producing N-desmethyltamoxifen (DMT) and 4-hydroxytamoxifen (4-OH-TAM) in both humans and rats. CYP 3A isoforms are the predominant subfamily involved in the formation of DMT and recent studies have shown that TAM induces hepatic forms of these enzymes. TAM’s inductive effect on gastrointestinal CYP 3A has not been previously reported. The current studies investigated TAM’s induction of CYP isoforms (3A and 2B) in female rat gastrointestinal and hepatic tissue at the mRNA, protein, and catalytic level. Since previous studies have not addressed whether TAM induction causes changes to the overall pharmacokinetics (PKs), a rat PK model was used to determine if TAM induced its own metabolism, and/or the metabolism of a CYP 3A substrate, midazolam (MDZ). Phenobarbital (PB) and/or dexamethasone (DEX) were used as positive controls for all studies. TAM significantly induced, or caused a trend towards induction of all studied parameters for hepatic CYP 3A and 2B, whereas intestinal CYP 3A and 2B analysis did not show significant induction by TAM at any level. A study evaluating time-dependent alterations in the PK profile of TAM showed no change in apparent oral clearance (Clapp) during two weeks of chronic dosing with TAM. However, the Clapp for MDZ was shown to trend towards an increase after two weeks of dosing with TAM, in a second PK study. These combined investigations suggest that TAM is an inducer of rat hepatic CYP 3A and 2B isoforms, and this agent has the potential of influencing the PK of coadministered 3A substrates.

Effects of benzodiazepine administration on A1 adenosine receptor binding in-vivo and ex-vivo

Abstract— The adenosine receptor has been implicated in the central mechanism of action of benzodiazepines. The specific binding of an A1‐selective adenosine antagonist radioligand, [3H]8‐cyclopentyl‐1,3‐dipropylxanthine, was measured in‐vivo in mice treated with alprazolam (2 mg kg−1, i.p.), lorazepam (2 mg kg−1, i.p.) and vehicle. Binding studies were performed in‐vivo and ex‐vivo in mice receiving continuous infusion of alprazolam (2 mg kg−1 day−1), lorazepam (2 mg kg−1 day−1) and vehicle by mini‐osmotic pumps for 6 days. Continuous infusion of alprazolam and lorazepam significantly decreased specific binding by 34 and 53%, respectively, compared with vehicle treatment (P < 0·01). Single doses of alprazolam and lorazepam induced a similar trend in specific binding in‐vivo (P = 0·07). There were no alterations in A1 ‐receptor density (Bmax) or affinity (Kd) in cortex, hippocampus or brainstem in ex‐vivo studies. Benzodiazepine treatment may diminish A1‐ receptor binding in‐vivo by inhibiting adenosine uptake or by direct occupancy of the A1 adenosine receptor recognition site.